Peptides and antibodies to muc 1 proteins

ABSTRACT

The invention relates to methods of inhibiting proliferation or growth of tumor cells and/or inducing cell death relates to the use of antibodies, hybridomas and pharmaceutical compositions containing same, for inhibiting growth or proliferation and inducing death in epithelial, colon, lung, breast and ovarian tumor cells or other cells which express MUC1 proteins.

FIELD OF THE INVENTION

The invention relates to methods of inhibiting proliferation or growthof tumor cells and/or inducing cell death in cancer cells. Thisinvention relates to the use of antibodies, hybridomas andpharmaceutical compositions containing same, for inhibiting growth orproliferation and inducing death in epithelial, colon, lung, breast andovarian tumor cells or other cells which express MUC1 proteins.

BACKGROUND OF THE INVENTION

Tumor markers are molecules that are associated with the transformationof a normal cell into a malignant cell. Tumor markers are either alteredproteins which are different from the proteins expressed in normal cellsor over-expression of proteins that are not expressed, or slightlyexpressed in normal cells.

Mucins are high molecular weight glycoproteins, which are produced bynormal epithelial cells. MUC1 is one of the four mucins known to datethat are transmembrane molecules and while its function in adult lifemaybe lubrication, in fetal life development it is thought to play animportant role in forming the lumen of the duct by keeping apart cellslocated opposite one another. The MUC1 gene was also shown to beexpressed in hemopoietic tissues. It was found that MUC1 (also calledH23-Ag, episialin, PEM-Polymorphic Epithelial Mucin, MCA-MammaryCarcinoma Antigen and EMA Epithelial Membrane Antigen) expression iselevated (10-50 fold) in breast cancer cells in comparison to normalresting mammary secretory epithelial cells. Moreover,immunohistochemical analyses using the H23 mAb (which recognizes MUC1),revealed that MUC1 is expressed in 91% of the breast cancer tissues and100% of breast cancer metastases, whereas the non-malignant tissues werenegative for the H23 mAb staining (1). Elevated levels of the MUC1protein in serum and body fluids were reported in 7%, 17%, 64% and 67%of breast cancer patients presenting with stages I to IV, respectively.In addition, elevated levels of circulating MUC1 may be associated witha poor

It was shown that MUC1 is over expressed in epithelial cancers otherthan breast cancer. MUC1 was shown to be over expressed in epithelialovarian cancer cells as well as in all types of lung cancer cells andother cancers (2).

In malignancy, the MUC1 oligosacharide chains are shorter and less densecomparing to MUC1 in normal cells. This results in the exposure of newepitopes of the core protein in the cancer-associated mucin.

Isolation of MUC1 cDNAs revealed several protein isoforms: the MUC1/REP,MUC1/SEC MUC1/Y and MUC1/X proteins. A short description of four of theabove-mentioned isoforms that are connected with the present inventionis given below:

The MUC1/REP isoform is a transmembrane protein that contains: a largeextracellular domain consisting of a heavily glycosylated 20 amino acidrepeat motif. The number of these repeats varies from 20 to 100 and thusis named a VNTR (Variable Number of Tandem Repeats); a transmembranedomain, which consists of a 28 amino acid hydrophobic sequence, and a 72amino acid cytoplasmic domain. During the biosynthesis of the MUC1/REPprotein it undergoes a proteolytic cleavage event. The cleavage takesplace within the conserved sequence IKFRPGSVVV that is contained withinthe extracellular domain. Intriguingly, this cleavage site resideswithin a previously identified module, designated the “SEA” module (3),found in a number of highly O-linked glycosylated proteins that areinvariably linked in one way or another to the cell membrane. The SEAmodule functions not only as a site for proteolytic cleavage, but alsofor subsequent re-association of the subunits. Consequently, theMUC1/REP protein is presented on the cell surface as a heterodimer whichis composed of a large extracellular subunit (containing the repeatarray) linked by non-covalent, SDS sensitive bonds to a smallercell-anchored subunit which consists of a small extracellular fragmentfollowed by the transmembrane and cytoplasmic domains. The largeextracellular subunit can disconnect and reconnect with the smallextracellular fragment of the cell-anchored subunit.

The MUC1/Y isoform is a transmembrane protein that containstransmembrane and cytoplasmic domains identical to those of MUC1/REPprotein. Unlike MUC1/REP, due to a differential splicing event thatutilizes splicing sites located upstream and downstream to the repeatarray, MUC1/Y protein is devoid of both the tandem repeat array and itsflanking regions. Expression of MUC1/Y was demonstrated in various humansecretory epithelial tumors. MUC1/Y was found to be expressed on thecell surface of various human epithelial tumor cells but is notdetectable in the adjacent normal tissue.

The MUC1/X isoform (4) is a transmembrane protein that containstransmembrane and cytoplasmic domains identical to those of MUC1/REPprotein. Unlike MUC1/REP, due to a differential splicing event thatutilizes splicing sites located upstream and downstream to the repeatarray, MUC1/X protein is devoid of both the tandem repeat array and itsflanking regions.

The MUC1/SEC isoform: This isoform is generated by an alternativesplicing mechanism. It is a secreted protein since it lacks thehydrophobic region that can attach the protein to the cell membrane. ItsN-terminal sequences are identical to those of the MUC1/REPextracellular domain. Furthermore it has been shown that the solublesecreted MUC1/SEC protein may bind specifically to the extracellulardomain of the MUC1/Y protein (5).

Thus, it will be highly advantageous to develop a ligand which binds toa specific epitope in the MUC1 proteins and, more particularly, to aspecific extracellular epitope in the MUC1/REP or MUC/Y proteins thatwill dramatically inhibit the proliferation or growth of cells, and willinduce death in cells, such as, cancer cells and in particular cellswhich over express MUC1 proteins.

SUMMARY OF THE INVENTION

In one embodiment, this invention provides methods for inhibiting cellproliferation or growth and/or inducing cell death in cancer cells andin particular in epithelial, breast, colon, lung and ovarian tumor cellswhich express MUC1 proteins.

In one embodiment, the invention provides a method of selectivelyinhibiting the proliferation or cell growth of cells, comprising thestep of administering to a subject, an effective amount of a ligandwhich specifically binds to an epitope in the extracellular region of atransmembrane isoform of MUC1 protein, thereby selectively inhibitingproliferation or growth of such cells.

In one embodiment, the invention further provides a method of inducingcell death comprising the step of administering to a subject, aneffective amount of a ligand which specifically binds to an epitope inthe extracellular region of a transmembrane isoform of MUC1 protein,thereby selectively inducing cell death.

In one embodiment, the invention provides a method of selectivelyinhibiting cell proliferation or cell growth comprising the step ofcontacting a cell which expresses MUC1 protein isoform with an effectiveamount of a ligand which specifically binds to an epitope in theextracellular region of the transmembrane isoform of MUC1 protein,thereby selectively inhibiting the cell proliferation or cell growth.

The invention provides a method of inducing cell death comprising thestep of contacting a cell which expresses MUC1 protein isoform with aneffective amount of a ligand which specifically binds to an epitope inthe extracellular region of the transmembrane isoform of MUC1 protein,thereby selectively inducing cell death.

In one embodiment, the invention further provides a method of treating asubject with a disease involving pathological proliferation of cellscomprising the step of administering to a subject, an effective amountof a ligand which specifically binds to an epitope in the extracellularregion of a transmembrane isoform of MUC1 protein, thereby treating thedisease.

In one embodiment, the invention further provides a method of treating asubject with a disease involving pathological proliferation of cellscomprising the step of administering to a subject, an effective amountof a peptide which comprises an amino acid sequence corresponding to theextracellular region of a transmembrane isoform of MUC1 protein, so asto induce an increase in the level of antibodies specific for saidpeptide in the subject, thereby treating the disease.

In one embodiment, the invention further provides an isolated antibodywhich specifically binds to an epitope in the extracellular region of anisoform of MUC1 protein wherein said epitope is located within a 59amino acid sequence according to the amino acid sequence of SEQ ID No.1.

In one embodiment, the invention further provides a pharmaceuticalcomposition comprising an effective amount of a ligand, whichspecifically binds to a MUC1 protein isoform, and a pharmaceuticallyacceptable carrier.

In one embodiment, the invention further provides a hybridoma cellproducing monoclonal antibody that binds to an epitope in theextracellular region of an isoform of MUC1 protein. In one embodimentthe epitope is located within a 59 amino acid sequence according to theamino acid sequence of SEQ ID No.1 and is located directly N′-terminalto the transmembrane domain of the protein.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be understood and appreciated more fully fromthe following detailed description taken in conjunction with theappended drawings in which:

FIG. 1 demonstrates expression of MUC1 proteins in epithelial celltransfectants. HBL-100 cells were transfected either with (A) theexpression vector harboring the MUC1/Y cDNA (H/Y2) or (B) with theselection plasmid pSV2neo (H/N). The expression of the various MUC1isoforms was analyzed by incubating the different transfectants usingthe (A1, B1), anti-MUC1/Y specific mAb BOS6E6/2 or (A2, B2)anti-tandem-repeat mAb H23.

FIG. 2 shows the different MUC1 protein isoforms and the location of theepitopes of the monoclonal specific antibodies.

FIG. 3A-D show the effect of anti-MUC1 monoclonal antibodies on thegrowth of H/Y2 cells (A) using MTT assay and (B-D) by assessing the cellmorphology by microscope.

FIG. 4 shows that the effect of BOS7D10 on cell growth is dependent onserum concentration. H/Y2 cells were exposed to medium supplemented with0.1% (lanes 6;12); 0.2% (lanes 5; 11); 0.3% (lanes 4;10); 1% (lanes3;9); 3% (lanes 2;8); and 10% (lanes 1;7) FCS. The effect of thedifferent mAb on the H/Y2 cells was analyzed by MTT in order to measurethe inhibitory effect of mAbs on cell proliferation.

FIG. 5 shows the sequence of MUC1/REP protein.

FIG. 6 demonstrates the 59 amino acid sequence that includes the epitoperecognized by the BOS7D10 monoclonal antibodies.

DETAILED DESCRIPTION OF THE INVENTION

MUC1 is one of the four mucins which are transmembrane glycoproteinsproduced by normal epithelial cells. As was described before, itsexpression is markedly increased in all human breast cancer and otherepithelial cancer cells. The sequence of MUC1 is demonstrated on FIG. 5.

The terms “specific binding” or “specifically bind” hereinabove in thespecifications and in the claims section refer to the interactionbetween a protein, a peptide, an agonist, an antibody, or an antagonist.The interaction is dependent upon the presence of a particular structureof the protein, e.g., the antigenic determinant or epitope, recognizedby the binding molecule.

In another embodiment the ligand can be an antibody, antibody fragments,an antagonist, an agonist or a peptide.

The term “contacting the cell” refers herein above in the specificationand in the claims refers to administering the cell, or exposing the cellto the ligand, which binds to a specific epitope on MUC1/Y or MUC1/X orMUC1/REP by methods of direct application, which are known in the art.The ligand can be applied before or after the seeding of the cells.

The term “antagonist” refers to a molecule which, when bound to theepitope, decreases the amount or the duration of the effect of thebiological or immunological activity of epitope. Antagonists may includeproteins, nucleic acids, carbohydrates, antibodies, antisense or anyother molecules which decrease the effect of MUC1 epitope on cell growthor cell viability.

“Antibody fragments” comprise a portion of a full length antibody,generally the antigen binding or variable region thereof. Examples ofantibody fragments include Fab, Fab′, F(ab′)₂, and Fv fragments;diabodies; linear antibodies; single-chain antibody molecules; andmultispecific antibodies formed from antibody fragments.

The term “agonist” refers hereinabove in the specifications and in theclaims to any molecule which, when bound or activate the epitope,increases the amount or the duration of the effect of the biological orimmunological activity of epitope. The molecule can be a peptide or anucleic acid construct.

As demonstrated herein, Applicants unexpectedly discovered the effect ofthe antibody BOS7D10 on cell growth and/or proliferation which isclearly exemplified in Example 2 and in FIG. 3. The effect of thisantibody and other antibodies to MUC1/Y and MUC1/SEC on cell viabilitywas assessed by MTT assay and by using a microscope, which are wellknown methods in the art. The effect of BOS7D10 which is directed to anepitope which is included in the 59 amino acid sequence as set forth onSEQ ID No.1, wherein the 59 amino acids sequence is located directlyN-terminal to the transmembrane domain of the MUC1/Y protein isoform andMUC1/X protein isoform and MUC1/REP protein isoform, was dramatic incomparison to other antibodies which bind to different epitopes on thesame protein.

The term “selectively inhibiting cell proliferation or cell growth” asused hereinabove in the specifications and in the claims, means killingor reducing growth i.e. differentiation, or proliferation i.e.propagation of pathologically proliferating cells without causingunacceptable killing or reduction of growth or proliferation of normalcells. In one embodiment, the killing or reducing growth i.e.differentiation, or proliferation i.e. propagation of pathologicallyproliferating cells is by at least 20%. The antibody described in theinvention was shown as a potent agent for inhibiting the proliferation(FIG. 3A).

“Selectively inducing cell death” refers hereinabove in thespecifications and in the claims, to cause a reduction in the viabilityof the pathological cells by at least 20%. The cell death can bedemonstrated by methods of assessing cell viability such as morphologyor use of different dyes such as MTT assay or Propidium Iodide (PI)staining, trypan blue assay, pre-cytolytic DNA fragmentation,alterations in membrane asymmetry, activation of apoptotic caspases andrelease of cytochrome C into the cytoplasm by mitochondria, degradationof actin filaments which are well known to someone who is skilled in theart.

There can be many processes by which cell death is achieved and some ofthese can lead to apoptosis. There is speculation that there are othercell death mechanisms other than apoptosis but regardless of how thecell arrives at death there are some characteristics of cell death. Oneof these is the absence of metabolism and another is the denaturation ofenzymes. In either case vital stains will fail to stain these cells.These endpoints of cell death have been long understood and predate thecurrent understanding of the mechanisms of cell death. Furthermore,there is the distinction between cytotoxic effects where cells arekilled and cytostatic effects where the proliferation of cells areinhibited. According to the data shown in the Examples section, theantibody of the invention is efficient as a cytotoxic agent as well as acytostatic agent and is considered as an apoptotic antibody.

The “cell” of the present invention refers in the specification and inthe claims section to a pathologically proliferating cell such as cancercells which according to another embodiment of the invention expressMUC/1 proteins. In another embodiment the MUC1 proteins are overexpressed.

The cancer cell may be, without being limited, an epithelial cell, abreast cancer cell, a colon cancer cell, a lung cancer cell or any cell,which expresses MUC1 protein isoforms. MUC1 protein isoforms comprises,without being limited, protein isoforms such as MUC1/Y, MUC1/X, MUC1/REPand MUC1/SEC proteins. “Isoforms” are proteins that are translated fromthe same gene but due to post translational or post transcriptionalmodifications, may contain different number of amino acids and/ordifferent additional groups and/or have a different molecular structure.

MUC1/Y, MUC1/X and MUC1/REP have identical transmembrane and cytoplasmicdomains. More important, the domain located N′-terminal to thetransmembrane domain is identical in these two isoforms (red box in FIG.2) Thus, the antibodies of the invention can bind to an epitope, whichexist on both protein isoforms.

The term “epitope” refers to the particular part of the antigen makescontact with a particular antibody. According to an embodiment of theinvention, the epitope length is 4-12 amino acids. In another embodimentthe epitope length is 5-10 amino acids, in yet another embodiment theepitope length is 6-8 amino acids. The epitope sequence is includedwithin the 59 amino acid sequence as set forth in SEQ ID No. 1 andbelow: (N-terminus) SVV VQLTLAFREG TINVHDVETQ FNQYKTEAAS RYNLTISDVSVSDVPFPFSA QSGAGV (C-terminus)

In another embodiment the epitope is located in the 15 amino acidsequence that resides at the N-terminal portion of the 59 amino acidsegment which is located directly N-terminal to the transmembrane domainof the MUC1/Y, MUC1/X and MUC1/REP proteins. The epitope is located inthe extracellular region of the transmembrane isoform of the MUC1/Y,MUC1/X and MUC1/REP proteins.

The term “extracellular region” refers hereinabove in the specificationand in the claims to a strech of a variable number of amino acidslocated directly N-terminal to the transmembrane domain and appears inthe extracellular environment.

The term “transmembrane” refers hereinabove in the specification and inthe claims to a strech of amino acids, mostly hydrophobic, locatedwithin the membrane, N-terminal to the cytoplasmic domain and C-terminalto the extracellular domain of the protein. This domain enables theanchor of the protein to the membrane.

The term “antibody” refers hereinabove in the specification and in theclaims to an immunoglobulin whether natural or partly or whollysynthetically produced. The antibodies can be human or animalicantibodies. The term also covers any polypeptide or protein having abinding domain which is, or is homologous to, an antibody bindingdomain. These can be derived from natural sources, or they may be partlyor wholly synthetically produced. Examples of antibodies are theimmunoglobulin isotypes and their isotypic subclasses; fragments whichcomprise an antigen binding domain such as Fab, scFv, Fv, dAb, Fd; anddiabodies. As is examplified on Example 2, the antibody BOS7D10 showed apotent inhibitory effect on cell growth as well as a potent cell deathactivity on MCF7.

It is possible to take monoclonal and other polyclonal antibodies anduse techniques of recombinant DNA technology to produce other antibodiesor chimeric molecules which retain the specificity of the originalantibody. Such techniques may involve introducing DNA encoding theimmunoglobulin variable region, or the complementarity determiningregions (CDRs), of an antibody to the constant regions, or constantregions plus framework regions, of a different immunoglobulin. See, forinstance, EP-A-184187, GE 2188638A or EP-A-239400. As antibodies can bemodified in a number of ways, the term “antibody” should be construed ascovering any specific binding member or substance having a bindingdomain with the required specificity. Thus, this term covers antibodyfragments, derivatives, functional equivalents and homologues ofantibodies, including any polypeptide comprising an immunoglobulinbinding domain, whether natural or wholly or partially synthetic.Chimeric molecules comprising an immunoglobulin binding domain, orequivalent, fused to another polypeptide are therefore included. Cloningand expression of chimeric antibodies are described in EP-A-0120694 andEP-A-0125023.

It has been shown that fragments of a whole antibody can perform thefunction of binding antigens. Examples of binding fragments are (i) theFab fragment consisting of VL, VH, CL and CHI domains; (ii) the Fdfragment consisting of the VH and CHI domains; (iii) the Fv fragmentconsisting of the VL and VH domains of a single antibody; (iv) the dAbfragment (8) which consists of a VH domain; (v) isolated CDR regions;(vi) F(ab′)2 fragments, a bivalent fragment comprising two linked Fabfragments (vii) single chain Fv molecules (scFv), wherein a VH domainand a VL domain are linked by a peptide linker which allows the twodomains to associate to form an antigen binding site (9-10). (viii)bispecific single chain Fv dimers (PCT/US92/09965) and (ix) “diabodies”,multivalent or multispecific fragments constructed by gene fusion(WO94/13804; (11). Diabodies are multimers of polypeptides, eachpolypeptide comprising a first domain comprising a binding region of animmunoglobulin light chain and a second domain comprising a bindingregion of an immunoglobulin heavy chain, the two domains being linked(e.g. by a peptide linker) but unable to associate with each other toform an antigen binding site: antigen binding sites are formed by theassociation of the first domain of one polypeptide within the multimerwith the second domain of another polypeptide within the multimer(WO94/13804).

One of the potential benefits of monoclonal antibodies with respect tothe treatment of cancer is their ability to specifically recognizesingle antigens. It was thought that in some instances cancer cellspossess antigens that were specific to that kind of transformed cell. Itis now more frequently believed that cancer cells have few uniqueantigens, rather, they tend to over-express a normal antigen such asMUC1 proteins or express fetal antigens. Nevertheless, the use ofmonoclonal antibodies provided a method of delivering reproducible dosesof antibodies to the patient with the expectation of better responserates than with polyclonal antibodies.

As exemplified in Example 3, a surprising, unexpected correlation hasbeen found between the effect of the antibodies on the interaction ofMUC1/SEC and MUC1/Y and their effect on cell death. An antibody that waspotent in inducing cell death was also potent in inducing interactionbetween the isoforms and vice versa (see table 1). This surprisingcorrelation may be related to the mechanism of action of the antibody ininducing cell death.

The invention further provides an isolated antibody, which binds to thedescribed epitope. The antibody can be a monoclonal antibody, asynthetic antibody, a polyclonal antibody or a chimera e.g., mousevariable regions joined to human constant regions.

In another embodiment this invention provides a method of treating asubject with a disease involving pathological proliferation of cellscomprising the step of administering to a subject in need an effectiveamount of a peptide comprising an amino acid sequence corresponding tothe extracellular region of a transmembrane isoform of MUC1 protein,said peptide is for example a 59 amino acid sequence of SEQ ID No. 1which is located directly N′-terminal to the transmembrane domain of theMUC1 protein. According to the invention, said peptide induces anincrease in the level of antibodies specific for said peptide in thesubject. The term “induces an increase in the level of antibodiesspecific for said peptide” refers to an increase in the level ofspecific antibodies directed against said peptide above the basal levelof said antibodies in non-treated subject.

The amino acid sequence of the region that includes the epitope of theantibody is shown in FIG. 6. BOS7D10 has been deposited at the AmericanType Culture Collection, Rockville, Md. USA under Accession numberATCC—accession No. XXXX.

The terms “amino acid” or “amino acid sequence” refer to anoligopeptide, peptide, polypeptide, or protein sequence, or a fragmentof any of these, and to naturally occurring or synthetic molecules.

The invention further provides a pharmaceutical composition comprisingan amount of a ligand (such as an antibody, a peptide or an antagonist),which specifically binds to MUC1 protein isoforms and a pharmaceuticallyacceptable carrier.

The term “pharmaceutically acceptable carrier” refers to a carrier or adiluent that does not cause significant irritation to an organism anddoes not abrogate the biological activity and properties of theadministered compound.

The invention methods and ligands can be further extended to treatmammals and especially humans with diseases involving pathologicalproliferation of mammalian cells such as cancer. To “treat” refers to:(i) preventing a disease, occurring in an animal that may be predisposedto the disease, disorder and/or condition, but has not yet beendiagnosed as having it; (ii) inhibiting the disease i.e., arresting itsdevelopment; and (iii) relieving the disease i.e., causing regression ofthe disease.

Thus, the invention further provides a method of inhibiting pathologicalproliferation of mammalian cells or cell growth comprising the step ofadministering to a subject in need, an effective amount of a ligandwhich specifically binds to an epitope in the extracellular region of atransmembrane isoform of MUC1 protein thereby selectively inhibitingpathological proliferation or growth of mammalian cells.

In another embodiment the invention provides a method of inducing celldeath comprising the step of administering to a subject in need, aneffective amount of a ligand, which specifically binds to an epitope inthe extracellular region of a transmembrane isoform of MUC1 proteinthereby inducing cell death.

The step of administering may involve either direct injection of theligand with a pharmaceutically accepatble carrier that will be targetedto the target cells. Alternatively, a prodrug may be used that will beactivated only in the required target cells either by different enzymesor by different chemical-physical conditions.

Liposomes have been used successfully to administer medications tocancer patients, and have been shown to be useful clinically in thedelivery of anticancer drugs such as doxorubicin, daunorubicin, andcisplatinum complexes (12-14).

Similarly, micelles have also been used to deliver medications topatients, (15) and micelles have been used as drug carriers and fortargeted drug delivery, (16-17) including cancer medications, (18-19).

If the antibody or composition is suitable for oral administration theformulation may contain, in addition to the active ingredient, additivessuch as: starch e.g. potato, maize or wheat starch or cellulose orstarch derivatives such as microcrystalline cellulose; silica; varioussugars such as lactose; magnesium carbonate and/or calcium phosphate. Itis desirable that, if the oral formulation is for administration it willbe well tolerated by the patient's digestive system. To this end, it maybe desirable to include in the formulation mucus formers and resins. Itmay also be desirable to improve tolerance by formulating the antibodyor compositions in a capsule which is insoluble in the gastric juices.It may also be preferable to include the antibody or composition in acontrolled release formulation.

If the antibody or composition is suitable for rectal administration theformulation may contain a binding and/or lubricating agent; for examplepolymeric glycols, gelatins, cocoa-butter or other vegetable waxes orfats.

The pharmaceutical compositions utilized in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, or rectalmeans. Alternatively, the antibody or composition may be in dry form,for reconstitution before use with an appropriate sterile liquid.

In addition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically acceptable carriers comprisingexcipients and auxiliaries, which facilitate processing of the activecompounds into preparations, which can be used pharmaceutically. Furtherdetails on techniques for formulation and administration may be found inthe latest edition of Remington's Pharmaceutical Sciences (MaackPublishing, Easton Pa.).

Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions, and the like,for ingestion by the patient.

Pharmaceutical preparations for oral use can be obtained throughcombining active compounds with solid excipient and processing theresultant mixture of granules (optionally, after grinding) to obtaintablets or dragee cores. Suitable auxiliaries can be added, if desired.Suitable excipients include carbohydrate or protein fillers, such assugars, including lactose, sucrose, mannitol, and sorbitol; starch fromcorn, wheat, rice, potato, or other plants; cellulose, such as methylcellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums, including arabic and tragacanth; andproteins, such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, and alginic acid or a salt thereof, such as sodiumalginate.

Dragee cores may be used in conjunction with suitable coatings, such asconcentrated sugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound, i.e., dosage.

Pharmaceutical preparations, which can be used orally, include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating, such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with fillers or binders, such aslactose or starches, lubricants, such as talc or magnesium stearate,and, optionally, stabilizers. In soft capsules, the active compounds maybe dissolved or suspended in suitable liquids, such as fatty oils,liquid, or liquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations suitable for parenteral administration maybe formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks' solution, Ringer's solution, orphysiologically buffered saline. Aqueous injection suspensions maycontain substances, which increase the viscosity of the suspension, suchas sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally,suspensions of the active compounds may be prepared as appropriate oilyinjection suspensions. Suitable lipophilic solvents or vehicles includefatty oils, such as sesame oil, or synthetic fatty acid esters, such asethyl oleate, triglycerides, or liposomes. Non-lipid polycationic aminopolymers may also be used for delivery. Optionally, the suspension mayalso contain suitable stabilizers or agents to increase the solubilityof the compounds and allow for the preparation of highly concentratedsolutions.

For topical or nasal administration, penetrants appropriate to theparticular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

After pharmaceutical compositions have been prepared, they can be placedin an appropriate container and labeled for treatment of an indicatedcondition.

Pharmaceutical compositions suitable for use in the invention includecompositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays, e.g., of neoplastic cells or inanimal models such as mice, rats, rabbits, dogs, or pigs. An animalmodel may also be used to determine the appropriate concentration rangeand route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.Effective dose is determined also in clinical trials in humans.

The ligand of the invention is capable of promoting the interaction ofMUC1/Y with MUC1/SEC. The change in morphology was observed in FIG. 3B-Dand might be related to the interaction of these two isoforms, whereinMUC1/SEC serves as a ligand to MUC1/Y which behaves as the receptor.

The term “cancer” is interpreted broadly. The ligand, such as antibodyor antagonist, of the invention can be “anti-cancer agent”, which termalso encompasses “anti-tumor cell growth agent” and “anti-neoplasticagent”. For example, the methods of the invention are useful fortreating cancers by inducing cell death or by inhibiting thepathological cell proliferation/or growth.

The invention further provides a hybridoma cell producing monoclonalantibody that binds to an epitope in the extracellular region of anisoform of MUC1 protein.

The hybridomas producing the monoclonal antibodies of the presentinvention are produced following the general procedures described byKohler and Milstein, Nature, 256, p. 495 (1975). In that procedure,hybridomas are prepared by fusing antibody producing cells (typicallyspleen cells of mice previously immunized with a mucin antigen source)to cells from an immortal tumor cell line using somatic cellhybridization procedures. The agents used for immunization of animals(“immunogens”) to induce production of antibodies to mucin antigens.

For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others may be immunized by injectionwith the relevant epitope or with any fragment or oligopeptide thereof,which has immunogenic properties. Depending on the host species, variousadjuvants may be used to increase immunological response. Such adjuvantsinclude, but are not limited to, Freund's, mineral gels such as aluminumhydroxide, and surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol.Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) andCorynebacterium parvum are especially preferable Freund's adjuvant.

The hybridomas resulting from the fusion process are allowed to grow.Thereafter, the resulting supernatants are screened using immunoassayprocedures to detect antibodies present in the supernatants capable ofbinding to the specific antigens. In other cases, supernatants werescreened for their ability to bind cultured cancer cells.

The antibodies are designed for therapeutic treatment of cancer inpatients. Thus, the antibodies can be naked antibodies. In anotherembodiment, the antibodies can be conjugated to a cytotoxic drug. Theterm cytotoxic drug refers to any agent which kills cells for example,without being limited, a radioactive isotope. The antibodies can be usedto target other molecules to the cancer cells. Examples for cytotoxicdrugs are without being limited bromodeoxyuridine (BUdR),5-iododeoxyuridine (IUdR), bromodeoxycytidine, fluorodeoxyuridine(FudR), hydroxyurea, cisplatin.

It will be appreciated that the present invention is not limited by whathas been described and that numerous modifications, all of which fallwithin the scope of the present invention, exist. For example, while thepresent invention has been described with respect to the testedantibody, it could be that other antibodies which overlap part of theepitope described will demonstrate similar effect.

It will be appreciated by persons skilled in the art that the presentinvention is not limited by what has been particularly shown anddescribed herein above.

EXAMPLES

Materials and Methods

Cell Lines

HBL-100—An epithelial cell line was obtained from primary cultures ofcells derived from an early lactation sample of human milk (20). Thecells contain a tandemly integrated SV40 genome.

HBUneo (H/N)—Stable transfectants generated by transfecting the cloningvector pSV2neo to the HBL-100 cells.

HBLIY2 (HN2)—Stable transfectant generated by co-transfecting theexpression plasmid harboring the MUC1/Y cDNA and the cloning vectorpSV2neo.

Media for Cell Culture Growth

The DMEM growth medium (Dulbecco's modified Eagle's medium) waspurchased from Biological Industries, Kibbutz Beit Haemek, Israel.

Media Supplements:

Antibiotics: 10 μg/ml Nystatin, 100 μg/ml Streptomycin, 100 μg/mlAmpicillin. (Biological industries, Kibbutz Beit Haemek, Israel).

4 mM L-Glutamine (Biological industries, Kibbutz Beit Haemek, Israel).Heat inactivated (56° C. for 30 min.) 10% fetal calf serum (FCS)(Biological industries, Kibbutz Beit Haemek, Israel).

The mixture Oxaloacetate (Sigma), Pyruvate (Sigma), Insulin (Sigma)(OPI) was added to the growth medium of the hybridoma cells.

Hybridoma S.M (serum free) medium—DMEM growth medium (Dulbecco'smodified Eagle's medium) was diluted 1:1 with the F12 medium (Biologicalindustries, Kibbutz Beit Haemek, Israel) and supplemented with theantibiotics (10?g/ml Nystatin, 100 μg/ml Streptomycin, 100 μg/mlAmpicillin), 4 mM L-Glutamine and “BIOGRO-2”, synthetic serum freemedium supplement. All the above materials were purchased fromBiological Industries, Kibbutz Beit Haemek, Israel.

Flow cytometry medium—DMEM supplemented with the antibiotics describedabove, 5% FCS and 0.01% Na-Azide.

Antibodies

H23—Monoclonal antibody that recognizes an epitope within the MUC1tandem repeat array thus, it can detect MUC1/REP and MUC1/SEC.

BOS6E6—Anti-MUC1/Y specific monoclonal antibody, that recognizes theMUC1/Y unique epitope.

BOS7D10, BOS10D2, BOS10B3—Monoclonal antibodies that recognize epitopeswithin the MUC1/Y extracellular domain.

SEC7H10—Anti MUC1/SEC specific mAb, that recognizes the MUC1/SECC-terminal eleven amino acids.

Cell Culture

Growing Cells in Culture:

Cell were cultured in 25 cm² or 75 cm² tissue culture flasks (FalconCostar) with 5 ml or 10 ml culture medium respectively and kept in anincubator, at 37° C., 95% air and 5% CO₂.

Harvesting and Seeding of Cells:

Harvesting of cells growing in monolayer was performed by addition of0.5-1 ml of Trypsin-EDTA solution to the flask for 1-5 minutes (celltype preference). Subsequently, an equal amount of 10% FCS rich growthmedium was added to neutralize enzyme activity. Cells were seeded intissue culture flask containing growth medium.

Cell Counting:

Cells were counted using a hemocytometer. Cells were re-suspended in amedium containing 0.08% Trypan blue in order to detect dead cells.

Cell Transfections.

Cell transfection was performed using the following commercialtransfection kit: DOTAP Transfection reagent kit (Roche diagnostics),Effectene Transfection Reagent (Qiagen) and LipofectAMINE Transfectionreagent (Gibco-BRL). The transfection protocols were performed accordingto the manufacturer manual.

Flow-Cytometry Analysis (FACS Analysis).

Cell samples (10⁶ cells/sample), were washed with flow cytometry mediumand incubated for 2 hours at 4° C., with primary antibody diluted inflow cytometry medium. Cells were washed with flow cytometry medium andincubated for 45 min at 4° C. with the secondary anti mouse-FITCconjugate antibody, (diluted 1:50 in Flow cytometry medium) followed byextensive washing and cells resuspended in PBS containing 0.01%Na-Azide. The samples were analyzed by FACS Analyser (BecktonDickenson). The flow cytometry results were analyzed using the “CellQuest” software.

MTT Proliferation Assay.

20 μl of 0.5% MTT (Sigma) solution (in phosphate saline buffer), wereadded to HBL-Y cells growing in 200 μl medium in 96 wells plate. Cellswere incubated with the MTT reagent for 2h at 37° C., medium was removedfrom the cells, and the MTT crystals were dissolved for 15 minutes in95% ethanol and plates were read by the ELISA reader at 592 nm.

MUC1/SEC-MUC1/Y Interaction Elisa Assays 96 Micro-Titer.

“Maxi Sorp” plates (NUNC) were coated over night at room temperaturewith 100 μl conditioned medium containing MUC1/SEC. Following coatingplates were washed twice with phosphate saline washing buffer containing0.05% Tween 20 (0.05%) pH 7.0 (neutralized with 1M HCl) and blocked for2 h at 35° C. with phosphate saline washing buffer supplemented with 10%FCS. Recombinant MUC1/Y diluted in phosphate saline washing buffersupplemented with 10% FCS was incubated in plates for 2 hr at 35° C.Following incubation the plate was extensively washed with phosphatesaline washing buffer, and incubated for additional 2 hr at 35° C. withbiotinylated 6E6/2 mAb diluted 1:300 in phosphate saline washing bufferfollowed by extensive wash with phosphate saline washing buffer, andincubation with Streptavidin-HRP (diluted 1:1000). The reaction wasdeveloped with the OPD substrate (Sigma). The reaction was stopped byaddition of 30 μl of 2M Sulfuric acid and read at 490 nm.

Vectors

pSV2neo—Vector encoding the neomycin resistance gene allowing stablytransfected eukaryotic cells to be selected using G418 (21)

pCL642-MUC1/Y—pCL642 coding for the MUC1/Y isoform (6).

DNA Ligation.

Reaction was performed by incubating the following mixture, at agradually decreasing temperature from 25° C. to 4° C. for over-night.DDW As required 10 × T4 reaction buffer (NEB) ×1 Plasmid DNA ˜100 ng DNAInsert 3 × molar access of plasmid T4 DNA ligase (NEB) 1 unit Totalvolume 20 μlPlasmid Preparation

Both small and large-scale plasmid preparation (Mini and Maxi preps.respectively) were performed according to Maniatis handbook (Smbrook etal., 1990).

DNA concentration was determined by translating optical density (OD)units at 260 nm according to the following formula: 1O.D_(260nm)=40 μg

EXAMPLE 1

Establishment of MUC1/Y Expressing Cells

To generate MUC1/Y expressing cells, an epithelial cell line, HBL-100,was co-transfected with an eukaryotic expression vector harboring theMUC1/Y cDNA and the selection plasmid pSV2neo. Control cells weregenerated by transfecting cells only with the neomycin-resistanceconferring plasmid. Following transfections, many control transfectants(H/N) were generated and a solitary clone (H/Y2), which consistentlyexpressed MUC1/Y protein. The expression was confirmed by flow cytometryanalyses, using the anti-MUC1/Y mAbs (FIG. 1).

As can be seen from the above flow cytometry analyses, both the H/Y2 andthe H/N transfectants express endogenous MUC1tandem-repeat-array-containing isoforms.

EXAMPLE 2

Effects of Monoclonal Antibodies Recognizing the MUC1/Y and MUC1/SECProteins on Epithelial Cell Viability.

A number of antibodies directed against the MUC1 protein have been used.The regions of their immunoreactivity within the MUC1 proteins have beendefined (FIG. 2).

To assess the effect that anti-MUC1/Y and anti MUC1/SEC monoclonalantibodies exert on human epithelial cell growth, MUC1/Y expressinghuman epithelial cell transfectants (H/Y2) were seeded in wells that hadbeen previously coated with serum-free hybridoma supernatants whichcontained either anti-MUC1/Y (four mAbs which recognize differentepitopes in the MUC1/Y extracellular domain) or an anti-MUC1/SECmonoclonal antibody.

A dramatic effect was observed when the cells were plated on wellscoated with the monoclonal antibody BOS7D10 (FIG. 3A columns 2 and 2′and panel C). The other three anti-MUC1/Y monoclonal antibodies werewithout any discernible effect (FIG. 3A columns 1 and 1′, 3 and 3′, 4and 4′, and panels B and C). The potent inhibitory effect of hybridomaBOS7D10 on cell growth was still observed following an eight-folddilution of the serum-free hybridoma supernatant and was only titratedout at a final sixteen-fold dilution.

Similar results were observed when the antibodies were tested with MCF7human breast cancer cells as the target cells. Also in this case, themonoclonal antibody BOS7D10 exerted a potent cell death activity on theMCF7 cells. The other antibodies were without any noticeable effect onthe cells.

As was demonstrated in FIG. 3D, microscope analysis revealed that thecells that were treated with BOS7D10 had a different appearance thancells that were treated with other antibodies (FIG. 3B,C). In fact theyexhibit a morphology of non-viable cells.

A “box” titration of the BOS7D10 hybridoma supernatant (FIG. 4) versusthe concentration of fetal calf serum demonstrated that the cell-growthinhibitory activity could be clearly observed when cells were grown at afinal serum concentration of 0.3% and was also discernible, albeit to alesser extent, at a 1% final serum concentration (FIG. 4). Growth ofcells at serum concentrations higher than 1% abrogated the growthinhibitory activity exerted by the BOS7D10 antibodies.

Interestingly, the monoclonal antibody SEC7H10, directed against theeleven C-terminal amino acids of the MUC1/SEC protein, also demonstratedgrowth-inhibitory-activity (FIG. 3A columns 5 and 5′). This growthinhibition was, however, considerably less than that exerted by BOS7D10.A control monoclonal antibody isotype matched for the BOS7D10 andSEC7H10 antibodies was without any effect (data not shown).

EXAMPLE 3

Effects of Anti-MUC1/Y and Anti-MUC1/SEC Antibodies on the Binding ofMUC1/Y and MUC1/SEC

In parallel to the effect of the anti-MUC1/Y and anti-MUC1/SECantibodies on epithelial cell growth, we analyzed whether theseantibodies could affect, in any way, the binding of MUC1/SEC for theMUC1/Y protein (see Methods for description of the assay).

The antibody BOS7D10, which recognizes an epitope in the extracellulardomain of MUC1/Y markedly enhanced the binding of MUC1/Y to the MUC1/SECprotein. The anti-MUC1/SEC antibody SEC7H10 also increased the binding,albeit to a lesser extent than BOS7D10 (see table 1). The otherantibodies did not effect the MUC1/Y MUC 1/SEC interaction in the sameway (table 1). Intriguingly, these results are in direct correlation tothe antibodies effect on cell growth. BOS7D10, which elicits the highestcell-growth inhibition, has also a potent effect on MUC1/Y MUC1/SECinteraction. Similar correlation was shown for SEC7H10. It appears thatantibodies that increase MUC1/Y-MUC1/SEC binding may elicit a negativegrowth signal. This conclusion tallies well with previous datasuggesting that MUC1/SEC binding to MUC1/Y may exert a growth-inhibitorysignal. Indeed addition of conditioned medium containing the MUC1/SECprotein to cells expressing the MUC1/Y protein reduced the growth rateof the latter cells as well as inducing morphological alteration ofcells (53) that was accompanied by actin filament degradation thatcorrrelates with apoptotic processes. Furthermore, coinoculation ofhighly tumorigenic mouse mammary tumor cells engineered to express theMUC1/Y protein with MUC1/SEC expressing cells markedly reduced the tumorgrowth of the injected cells (a reduction of 67%) suggesting that thepresence of MUC1/SEC within the microenvironment of the injected highlytumorigenic mouse mammary tumor cells diminishes their tumor-formingability TABLE 1 Effect of anti-MUC1 mAbs on the MUC1/SEC-MUC1/Yinteraction and on cell death BOS BOS BOS BOS SEC 7D10/4 10D2/36 6E6/210B3/71 7H10/5 Effect on the

N.E.

MUC1/SEC-MUC1/Y interaction Cell death effect ++ − − − +

The effect of anti-MUC1 mAbs on the MUC1/SEC-MUC1/Y interaction wasanalyzed by a sandwich ELISA assay (described in Methods).

REFERENCES

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21. southern P J and Berg P Transformation of mammalian cells toantibiotic resistance with a bacterial gene under control of the SV40early region promoter. J. Mol. Appl. Gent. 1, 32741 (1982). SEQ ID NO 1(N-terminus) SVV VQLTLAFREG TINVHDVETQ FNQYKTEAAS RYNLTISDVS VSDVPFPFSAQSGAGV (C-terminus):

1. A method of inhibiting mammalian cell proliferation or cell growthcomprising the step of administering to a subject in need, an effectiveamount of a ligand which specifically binds to an epitope in theextracellular region of a transmembrane isoform of MUC1 protein, therebyselectively inhibiting mammalian cell proliferation or cell growth.
 2. Amethod of inducing mammalian cell death comprising the step ofadministering to a subject in need, an effective amount of a ligandwhich specifically binds to an epitope in the extracellular region of atransmembrane isoform of MUC1 protein, thereby selectively inducingmammalian cell death.
 3. A method of treating a subject with a diseaseinvolving pathological proliferation of cells comprising the step ofadministering to a subject in need, an effective amount of a ligandwhich specifically binds to an epitope in the extracellular region of atransmembrane isoform of MUC1 protein, thereby treating the disease. 4.A method of selectively inhibiting cell proliferation or cell growthcomprising the steps of contacting a cell which expresses MUC1 proteinisoform with an effective amount of a ligand which specifically binds toan epitope in the extracellular region of the transmembrane isoform ofthe MUC1 protein, thereby selectively inhibiting the cell proliferationor cell growth.
 5. A method of selectively inducing cell deathcomprising the step of contacting a cell which expresses MUC1 proteinisoform with an effective amount of a ligand which specifically binds toan epitope in the extracellular region of the transmembrane isoform ofMUC1 protein, thereby selectively inducing cell death.
 6. The methodaccording to claims 1 to 5, wherein said cell is a cancer cell.
 7. Themethod according to claim 6, wherein said cancer cell is an epithelialcancer cell.
 8. The method according to claim 7, wherein said epithelialcancer cell is a breast cancer cell, an ovarian cancer cell, a lungcancer cell or a colon cancer cell.
 9. The method according to claims 1to 5, wherein said MUC1 isoform is MUC1/Y, MUC1/REP, or MUC1/X.
 10. Themethod according to claims 1 to 5, wherein said ligand is an antibody, apeptide, an antagonist, or an agonist.
 11. The method according toclaims 1 to 5, wherein said epitope is an amino acid sequence within a59 amino acid sequence as set forth in SEQ ID No.1.
 12. The methodaccording to claims 1 to 5, wherein said ligand is conjugated to acytotoxic drug.
 13. The method according to claim 10, wherein saidantibody is a monoclonal antibody, a synthetic antibody, a polyclonalantibody or a chimera.
 14. A method of treating a subject with a diseaseinvolving pathological proliferation of cells comprising the step ofadministering to a subject in need, an effective amount of a peptidewhich comprises an amino acid sequence corresponding to theextracellular region of a transmembrane isoform of MUC1 protein, so asto induce an increase in the level of antibodies specific for saidpeptide in the subject, thereby treating the disease.
 15. The methodaccording to claim 14 wherein said MUC1 isoform is MUC1/Y, MUC1/REP, orMUC1/X.
 16. The method according to claim 14 wherein said peptidecomprises an amino acid sequence within a 59 amino acid sequence as setforth in SEQ ID No.1.
 17. An isolated antibody which specifically bindsto an epitope in the extracellular region of a transmembrane isoform ofMUC1 protein.
 18. The isolated antibody according to claim 17, whereinsaid isoform of MUC1 protein is MUC1/Y protein, MUC1/REP protein orMUC1/X protein.
 19. The isolated antibody according to claim 17, whereinthe antibody is a monoclonal antibody, a synthetic antibody, apolyclonal antibody or a chimera.
 20. The antibody according to claim 17wherein said epitope is an amino acid sequence within a 59 amino acidsequence as set forth in SEQ ID No.1.
 21. A hybridoma cell producing amonoclonal antibody that binds to an epitope in the extracellular regionof a transmembrane isoform of MUC1 protein.
 22. The hybriodoma of claim21, wherein said isoform of MUC1 protein is MUC1/Y protein, MUC1/REPprotein or MUC1/X protein.
 23. A pharmaceutical composition comprisingan effective amount of a ligand which specifically binds to an epitopewithin a transmembrane isoform of MUC1 protein and a pharmaceuticallyacceptable carrier.
 24. The pharmaceutical composition of claim 23wherein said ligand is conjugated to a cell killing agent.
 25. Thepharmaceutical composition of claim 23, wherein said MUC1 isoform isMUC1/Y, MUC1/REP or MUC1/X.
 26. The pharmaceutical composition of claim23, wherein said epitope is an amino acid sequence within a 59 aminoacid sequence as set forth in SEQ ID No.1
 27. The pharmaceuticalcomposition of claim 23, wherein said ligand is an antibody, a peptide,an antagonist or an agonist.
 28. The pharmaceutical composition of claim27, wherein the antibody is a monoclonal antibody, a synthetic antibody,a polyclonal antibody or a chimera.